Study On Several Conotoxins And Antipyretic Analgesic Drugs Targeting CAV2.2 Channels Via GABA_B Receptor

?-Aminobutyric acid?GABA?is a type of inhibitory neurotransmitter.GABA_BR is widely distributed in the central nervous system and peripheral nervous system and is closely related to various diseases,such as pain,depression,cancer,and drug addiction.GABA_BR is composed of two subunits GABA_B1 and GABA_B2,which their heterogenous dilatation can form a functional GABA_BR.Particularly,the activated GABA_BR located in presynaptic neuron inhibits N-type calcium channels?Cav2.2?or P/Q?Cav2.1?and reduces calcium ions inward flows through coupling Gi/o-type proteins.To date,the GABA_BR agonist baclofen have been used to treat sputum,reduce alcohol addiction and relieve pain,but it has apparent side effects.Several?-conotoxin,such as Vc1.1,PeIA,et al.also target on the GABA_B receptor-mediated Cav2.2and neuronal nicotinic acetylcholine receptors?nAChRs?,and have significant analgesic activity.In the previous study,Vc1.1 and its mutants were synthesized and their analgesic activities were determined using a classical analgesic model:the rat partial sciatic nerve ligation model?PSL?.We found that several mutants display higher analgesic activities than that of Vc1.1.However,the inhibitory activity to nAChRs is lower and the exact target is unknown.Besides,the target of novel conopeptides we found is unknown.In the other hand,the antipyretic analgesics possesses the significantly different structure,but the reported mechanism is the inhibition of cyclooxygenase?COX?and reduction of the production of prostaglandin?PG?.We suspect that some antipyretic analgesics may have other targets,such as the GABA_BR-mediated Cav2.2.Therefore,in order to discover a new GABA_BR-mediated Cav2.2 inhibitor,we determined the inhibitory activities of the above-mentioned conotoxins and antipyretic analgesics on GABA_BR-mediated Cav2.2.Except for the above study,the Bt22.1 mutants were synthesized to determine its structural activity relationship.The main research results are as follows:?1?Twenty-one?-conotoxin and mutants were successfully synthesized,such as Vc1.1,Vc1.1[N9R],Benzoyl-Vc1.1[N9R],Bt22.1,Bt22.1[D1A]and so on.?2?The electrophysiological platform was setup to determine the inhibitions of conotoxins and compounds on GABA_BR-mediated Cav2.2.?3?The activities of Vc1.1[N9R],and Benzoyl-Vc1.1[N9R]on GABA_BR-mediated Cav2.2 were determined.The results showed that benzoyl-Vc1.1[N9R]displayed high inhibitory activity,the IC₅₀ was 0.2?0.1⁰.3?nM and wasten times lower than that of Vc1.1?2.4?0.8⁷.0?nM?,but the activity of Vc1.1[N9R]is similar to Vc1.1.Vc1.1[N9R],and Benzoyl-Vc1.1[N9R]were no active to Cav2.2 alone.?4?The inhibitory activity of Lt1.3-I and Lt1.3-II on GABA_BR-mediated Cav2.2 were determined.The results showed that Lt1.3-II with the disulfide bonds“1-3,2-4”displayed high activity(IC₅₀=33.9?10.7¹07.1?nM),but Lt1.3-I with the disulfide bonds“1-3,2-4”was no activity on GABA_BR.Lt1.3-I and Lt1.3-II had no activity against Cav2.2 alone.?5?The inhibition of eight antipyretic analgesics,such as aspirin,ibuprofen,indomethacin,et al.,was determined using GABA_BR-mediated Cav2.2.The results showed that aspirin and diclofenac sodium had potent activity,the IC₅₀was 0.21?M?0.13⁰.34??M and 0.15?M?0.06⁰.37??M,respectively,but others had lower activity.In summary,the Vc1.1 mutant Benzoyl-Vc1.1[N9R]is 10-fold potent than that of Vc1.1 in the inhibition of GABA_BR-mediated Cav2.2,the conotoxin with the disulfide bond"C1-C4,C2-C3"and several antipyretic analgesics are first found to inhibit the GABA_BR-mediated Cav2.2.This study expands the target of conotoxins and antipyretic analgesics and provides a now clue to develop new pain drugs.